Vaccines and antibiotics help prevent or ameliorate many infectious diseases. Yet, natural evolution and engineering for bioterrorism purposes create novel biothreats for which novel vaccines may represent the most potent countermeasures. We now know that vaccines act through dendritic cells (DCs), the initiators and controllers of immune effectors (T and B lymphocytes) differentiation. Just as lymphocytes are composed of different subsets, DCs comprise several subsets that differentially control lymphocyte function. Unfortunately, there is a shortage of reagents that permit to distinguish these subsets at various stages of differentiation. While it is now recognized that pathogens target DCs, it is not known which subsets are affected and how. Here, we will develop, validate and standardize assays, reagents and technologies for studies in human DC subsets in vitro and in vivo. Aim 1 will generate mAbs specific for human DC subsets at distinct stages of maturation and establish mRNA microarray biosignatures of DC subsets exposed in vitro to Category B-C pathogens. Aim 2 will generate mice with a full human immune system for testing vaccine potency, microbe pathogenicity and establish in vivo biosignatures of Category B-C pathogens. Aim 3 will establish assays for in vitro and in vivo analysis of how Category B-C pathogens alter biology of human DC subsets. Aim 4 will establish methodologies for in vivo analysis of how Category B-C pathogens alter interactions of human DC subset with immune effectors. Deliverables of this project include: 1) Assays such as the use of gene microarrays and multiplex flow cytometry to identify human biosignatures of Class A- C pathogens, 2) Reagents such as numerous pure human DCs subsets and novel mAbs that permit discrimination among DC subsets at each stage of differentiation, 3) Novel technologies such as the Humouse, an in vivo model of the human immune system, as well as assays to analyze how pathogens alter the presentation of antigens to human immune effectors. This Technical Development project will enhance our knowledge of human DC subsets and create tools for evaluation of human vaccines.